Previously, we confirmed that genetic elimination of the NR1 subunit of NMDA receptors in Ppp1r2-Cre/NR1 KO mutant mouse line begins at postnatal day 7 and is targeted to 40-50% of cortical and hippocampal GABAergic cells, primarily parvalbumin positive ones. We then conducted an extensive battery of behavioral tests and found that the mutants display the constellation of phenotypes from cellular, physiology to behavior levels, many of which may resemble human schizophrenia symptoms. Therefore, it provides strong experimental support for the long-standing hypothesis that corticolimbic NMDAR hypofunction is a primary site of schizophrenia pathogenesis (Belforte et al., 2010). What are the direct downstream events of NMDAR deletion in the cortical interneurons? We observed in the NR1 mutants Cre-targeted neuron-specific decreases in GAD67 levels, which is known to be neuronal activity-dependent. Therefore, it is likely that decreased GAD67 is a direct consequence of NMDAR hypofunction in the interneurons. We sought to assess the impact of GAD67 reduction on the behavior by generating two additional corticolimbic GABAergic neuron knockout mutants: Ppp1r2-Cre/ErbB4 KO mice and Ppp1r2-Cre/Gad1 KO mice. The reason why we chose ErbB4 was that this is selectively expressed in the PV-positive interneurons in the cortex and appears to facilitate GABA release. Thus, genetic ablation of ErbB4 could inhibit GABA release, mimicking the Gad67 reduction. Indeed, we found a reduction of GABA release, and furthermore a robust increase in the mean firing of somatosensory cortex principal neurons, suggesting cortical disinhibition. Thus, we thought ErbB4 KO mutants would be an ideal animal to test the impact of impaired cortical GABAergic inhibition on the behavior. Unexpectedly, we observed fairly normal behavior of the mutants: anxiety, locomotion, spatial working memory, prepulse inhibition (PPI) of acoustic startle response, and mating/nest building are all normal. In addition, we also found that synchronized activity as measured by cross-correlation analysis from the tetrode unit recording was normal in the ErbB4 mutants, which is in sharp contrast to the data from NR1 mutants. Since ErbB4-deleted interneurons display intact NMDA currents, we expect that other downstream pathways of NR1 deletion, rather than impaired GABAergic inhibition, could disturb the synchronized activity in the cortex of the NR1 KO mutants, which in turn fails in the proper information processing, leading to the behavioral deficits. To directly assess the impact of GAD67 reduction, we have also generated and analyzed Ppp1r2-Cre/Gad1 heterozygous KO and homozygous KO mutant mice. We did not detect any behavioral abnormality in the heterozygous Gad1 KO mutants. In patients with schizophrenia, it was reported that GAD67 mRNA is not detectable in 50% of cortical interneurons. Thus, we also tested Ppp1r2-Cre/Gad1 homozygous KO mutants in which GAD67-IR is ablated from 50% of cortical and hippocampal interneurons, and we observed significantly distinct behavioral phenotypes. Preliminary results showed normal spatial working memory and PPI but impaired nest building behavior. A more robust phenotype is anhedonia, as measured by female urine sniffing test and running wheel test. The mutants also showed obesity, which is often observed in major depressive disorder patients. These results suggest that a robust reduction in GABA leads not to the schizophrenia-like phenotype seen in the NR1 mutants but rather to a significantly distinct domain of neuropsychiatric illnesses, perhaps more consistent with mood disorders. The in-depth analysis of Ppp1r2-Cre/Gad1 homozygous KO mutants is a topic for the next fiscal year.
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